PROJECT SUMMARY The goal of this proposal is to understand the discrete roles of the highly druggable targets, ORAI proteins in T cells for potential therapeutic exploitation. T cells require high and sustained levels of Ca2+ signaling for activation. Ca2+ signaling triggered by T cell receptor stimulation is primarily mediated by CRAC (Ca2+ release- activated Ca2+) channels that consist of the pore subunits, ORAI proteins. ORAI family members, ORAI1, ORAI2, and ORAI3, share a similar structure and can mediate CRAC currents but display distinct expression patterns and biophysical properties. Orai1 knockout (KO) effector T cells exhibit a reduction in Ca2+ entry, cytokine expression, and pathogenicity but also showed significant residual activity (20-40% of WT cells). These observations suggest that either ORAI2 or ORAI3 also contributes to Ca2+ entry in effector T cells. We find that, while deletion of Orai2 marginally influenced the function of effector T cells, Orai3 deletion substantially decreased Ca2+ entry and cytokine production. ORAI3 shares biophysical properties with ORAI1 but is unique in its resistance to inactivation by elevated intracellular Ca2+ levels and oxidative stress (e.g., reactive oxygen species, ROS) due to the absence of cysteine residues in its extracellular loop region. Based on these findings, the central hypothesis is that ORAI3 should be necessary for the in vivo effector T cell response in inflamed tissues. We propose to determine 1) the physiological outcomes of Orai3 deletion in effector T cells and 2) the distinctive role of ORAI3 homomultimeric channels in the effector T cell response. The proposed work is highly innovative because this study will determine the unknown role of ORAI3 in effector T cell responses. It will also define the distinct molecular identity of Ca2+ channels in T cell subsets. The idea to define the unique role of ORAI3 under oxidative stress at the inflamed tissue is conceptually innovative. This study to understand the physiological outcomes of blocking heteromeric or homomeric ORAI3 channels in the effector T cell response will have a significant positive impact on future therapeutic exploitation of the ORAI channels to treat autoimmunity or transplant rejection.